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Department of Cell Biology, Physiology and Immunology (V.M.N., J.M.C., R.F.-F., J.R., A.M., M.L.B., E.A., L.P., M.T.-S.), University of Córdoba, 14004 Córdoba, Spain; Institute of Endocrinology (P.M.), University of Milan, 20133 Milan, Italy; and Department of Physiology (S.T., R.N., M.J.V., C.D.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
Address all correspondence and requests for reprints to: Manuel Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology, and Immunology, Faculty of Medicine, University of Córdoba, Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain. E-mail: fi1tesem{at}uco.es.
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Introduction |
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In this context, an unexpected reproductive facet of the KiSS-1/GPR54 system has recently emerged (11, 13, 14). Thus, two independent reports recently provided conclusive evidence demonstrating that a number of point mutations and deletions of the GPR54 gene are found in patients suffering familiar forms of idiopathic hypogonadotropic hypogonadism (13, 14). This clinical syndrome was reproduced in mouse models carrying null mutations of the GPR54 gene (11, 14). Accordingly, the KiSS-1/GPR54 system was proposed to play a previously unsuspected, relevant role in the regulation of the development and/or function of the hypothalamic-pituitary-gonadal axis. Yet the regulatory mechanisms, site(s) of action, and biological effects of this system within the reproductive axis remain so far mostly unexplored. In fact, despite their central role in the control of reproductive function (15), detailed characterization of the role of the KiSS-1/GPR54 system in the neuroendocrine network governing the secretion of pituitary gonadotropins is still pending. In this sense, our recent data evidenced that hypothalamic expression of KiSS-1 and GPR54 genes is maximum at puberty and under the regulation of sex steroids and preliminarily demonstrated a potent LH-releasing activity of intracerebroventricular administration of KiSS-1 peptide in the rat (16). Likewise, Gottsch et al. (17) very recently reported the LH releasing action of KiSS-1 peptide in the mouse. In good agreement, data from mouse models carrying null mutations of the GPR54 gene demonstrated defective LH content and preserved response to LHRH at the pituitary (14). In this context, the present experimental work was undertaken to delineate the site(s) and mode of action of KiSS-1 in the control of pituitary LH secretion. To this end, the LH-releasing activity of KiSS-1 peptide was monitored in different in vivo and in vitro settings under different experimental conditions.
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Materials and Methods |
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Experimental designs
On the basis of our preliminary data showing a potent LH-releasing activity of KiSS-1 (16), in experiment 1 a detailed dose-response analysis of the effects of centrally administered KiSS-1 was conducted in pubertal (45 d old) male rats. This age point was selected, given the proposed key role of the KiSS-1/GPR54 system in the control of puberty onset (14). To this end, groups of males (n = 10) were implanted with intracerebroventricular (icv) cannulae, as described in detail elsewhere (18), and KiSS-1 was centrally injected over a range of doses from 1 nmol to 10 pmol in 10 µl. The highest dose was selected on the basis of our initial data on the ability of 1 nmol KiSS-1 to potently elicit LH secretion (16) and previous references testing the neuroendocrine actions of different centrally administered peptides (18, 19). Pair-aged males (n = 10) injected with vehicle (physiological saline; NaCl 0.9%) served as controls. Trunk blood samples were taken on decapitation of the animals at 15 and 60 min after KiSS-1 injection for hormone determination.
Because results from experiment 1 evidenced a potent stimulatory action of centrally injected KiSS-1 on LH secretion, the next experiments aimed at identifying the potential interplay between KiSS-1 and hypothalamic LHRH. Thus, in experiment 2, pubertal (45 d old) male rats (n = 10–12/group) were twice sc injected with a potent LHRH antagonist (5 mg/kg·24 h; two doses) to completely block endogenous LHRH actions. A detailed time-course analysis of serum gonadotropin levels after single administration of this dose of the antagonist has been previously reported, showing maximum suppression of circulating LH levels 24 h after injection of the analog (20). Vehicle-injected groups served as controls. Twenty-four hours after the last dose of the antagonist, the animals were icv injected with 1 nmol KiSS-1 or vehicle, and trunk blood samples were collected 15 min later. In addition, in experiment 3, the effects of combined administration of KiSS-1 and LHRH on LH secretion were evaluated. Pubertal (45 d old) male rats were simultaneously injected with LHRH (1 µg/rat, equivalent to 0.85 nmol; ip) and KiSS-1 (1 nmol/rat; icv). Vehicle-injected groups served as controls. Trunk blood samples were collected 15 min after administration of the peptides. Finally, the effects of centrally administered KiSS-1 peptide on LHRH gene expression were monitored in experiment 4. In detail, two experimental settings were used. Firstly, peripubertal (35 d old) male rats were chronically icv injected with KiSS-1 for 7 d at a dose of 1 nmol KiSS-1/12 h. On decapitation of the animals, hypothalamic samples were obtained 60 min after the last injection of KiSS-1. Second, early adult (60 d old) male rats were icv injected with a single dose of 1 nmol KiSS-1, and hypothalamic samples were collected 15, 60, and 180 min later. Hypothalamic tissue [including the preoptic area (POA)] was dissected out, as described in detail elsewhere (21), by a horizontal cut of about 2-mm depth with the following limits: 2 mm anteriorly from the optic chiasm, the posterior border of mamillary bodies, and the hypothalamic fissures. Hypothalamic samples were immediately removed on decapitation, frozen in liquid nitrogen, and stored at –80 C until processing for RNA analysis.
In the next series of experiments, the effects of systemic administration of KiSS-1 peptide on LH secretion were monitored. In experiment 5, KiSS-1 (7.5 nmol/rat; equivalent to 10 µg/animal) was administered ip to pubertal (45 d old) male rats, and trunk blood samples were obtained at 15 and 60 min. For comparative purposes, pair-aged males were icv injected with 1 nmol KiSS-1, as described in previous experiments. In addition, in experiment 6, the effect of iv injection of KiSS-1 on LH release was monitored in freely moving rats. To this end, groups of male rats (n = 6) were implanted with intracardiac cannulae, as described in detail elsewhere (22), and blood samples (250 µl) were taken every 15 min over a 240-min period. For proper handling, the animals were sampled four times before iv injection of KiSS-1 (7.5 nmol/rat) or vehicle. Finally, because data from the above experiments evidenced a clear stimulatory effect of systemic administration of KiSS-1, the ability of the peptide to modulate LH secretion directly at the pituitary level was addressed in experiment 7, using static incubations of pituitary tissue from pubertal (45 d old) male rats. Procedures for incubation of pituitary samples have been described in detail elsewhere (18, 22). Briefly, on decapitation of the animals, anterior pituitaries (n = 10–12 per group) were removed and placed in scintillation vials in a Dubnoff shaker at 37 C with constant shaking (60 cycles/min) under an atmosphere of 95% O2-5% CO2. After 1 h of preincubation, the media were replaced by either fresh medium alone (DMEM) or medium containing increasing doses of KiSS-1 (10–10, 10–8, 10–6 M). For comparative purposes, a group of pituitary samples was incubated with LHRH (10–8 M). Samples from the incubation media were collected at 60 and 180 min for hormone determinations.
In the final set of experiments, the potential interactions between central KiSS-1 and relevant neurotransmitters in the neuroendocrine control of LH secretion were explored. Thus, in experiment 8, the LH-releasing effect of central administration of KiSS-1 was monitored after blockade of NMDA and KA/AMPA receptors; i.e. the major ionotropic receptors for the excitatory amino acid (EAA) glutamate. To this end, groups of prepubertal (30 d old) male rats were ip treated with the NMDA receptor antagonist MK-801 (1 mg/kg) or the KA/AMPA receptor antagonist NBQX (0.5 mg/kg), in agreement with previous references (22, 23). Forty-five minutes after injection, 1 nmol KiSS-1 was icv injected, and trunk blood samples were taken on decapitation of the animals 15 min later. Similarly, NO dependency for the effects of KiSS-1 on LH secretion was assessed in experiment 9. Groups of prepubertal (30 d old) male rats were ip injected with the blocker of NO synthases NAME (40 mg/kg), as previously described (22). Forty-five minutes after injection, 1 nmol KiSS-1 was icv injected, and trunk blood samples were taken on decapitation of the animals 15 min later.
Hormone measurement by specific RIAs
Serum LH levels were measured in a volume of 25 µl using a double-antibody method and RIA kits kindly supplied by the National Institutes of Health (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program, Bethesda, MD). Rat LH-I-9 was labeled with 125I by the chloramine-T method, and the hormone concentrations were expressed using the reference preparation LH-RP-3 as standards. Intra- and interassay coefficients of variation were less than 8 and 10%, respectively. The sensitivity of the assay was 5 pg/tube. In addition, in selected serum samples (experiment 1), serum testosterone levels were determined using a commercial kit from MP Biomedicals (Costa Mesa, CA), following the instructions of the manufacturer. The sensitivity of the assay was 0.1 ng/tube, and the intraassay coefficient of variation was less than 5%. Accuracy of hormone determinations was confirmed by assessment of rat serum samples of known hormone concentrations used as external controls.
RNA analysis by semiquantitative RT-PCR
Total RNA was isolated from hypothalamic samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method (24). Expression of LHRH mRNA was assessed by RT-PCR, optimized for semiquantitative detection, using the primer pairs and conditions indicated in Table 1
. As internal control for reverse transcription (RT) and reaction efficiency, amplification of a 240-bp fragment of S11 ribosomal protein mRNA was carried out in parallel in each sample, as indicated in Table 1. For amplification of the targets, 2 µg of total RNA was used to perform RT-PCR in two consecutive separate steps. In addition, to enable appropriate amplification in the exponential phase for each target, PCR amplification of LHRH and RP-S11 transcripts was carried out in separate reactions with different number of cycles but using similar amounts of the corresponding cDNA templates, generated in single RT reactions, as previously described (16, 25). PCRs consisted in a first denaturing cycle at 97 C for 5 min, followed by a variable number of cycles of amplification defined by denaturation at 96 C for 30 sec, annealing at 58 C for 30 sec, and extension at 72 C for 1 min. A final extension cycle of 72 C for 15 min was included. Different numbers of cycles were tested to optimize amplification in the exponential phase of PCR. On this basis and our previously published work (16), the numbers of PCR cycles indicated in Table 1 were chosen for further semiquantitative analysis of LHRH and RP-S11 internal control. PCR-generated DNA fragments were resolved in Tris-borate buffered 1.5% agarose gels and visualized by ethidium bromide staining. Specificity of PCR products was confirmed by direct sequencing using a fluorescent dye termination reaction and an automated sequencer (Central Sequencing Service, University of Cordoba, Spain). Quantification of intensity of RT-PCR signals was carried out by densitometric scanning using an image analysis system (1-D Manager, TDI Ltd., Madrid, Spain), and values of the specific signal (LHRH) were normalized to those of internal controls to express arbitrary units of relative expression. In all assays, liquid controls and reactions without RT resulted in negative amplification.
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7- to 8-fold increase over controls). In contrast, although 60 min after injection all doses of KiSS-1 significantly stimulated LH levels, the magnitude of elevation of serum LH levels at this time point appeared as dose dependent, with peak values at 1 nmol and 500 pmol, and lower responses at 100 and 10 pmol KiSS-1 (Fig. 1). In good agreement with serum LH data, maximal responses to 1 nmol KiSS-1 at 60 min correlated with an extremely potent secretory effect in terms of testosterone release, whose serum levels were increased approximately 10-fold over control values (6.99 ± 0.85 vs. 0.75 ± 0.09 ng/ml in vehicle-injected animals).
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). Second, the effects of systemic administration of LHRH were compared with those of central injection of KiSS-1. As shown in Fig. 2B, administration of 1 µg/rat of LHRH maximally elicited LH secretion, with approximately 8- to 9-fold increase over controls. This response was mimicked by icv injection of 1 nmol (equivalent to 1.3 µg) KiSS-1 (8-fold increase over controls). Combined administration of LHRH and KiSS-1 was not able to further increase serum LH levels over those observed after individual administration of both peptides. Third, the effects of KiSS-1 on the expression levels of LHRH mRNA were monitored. Chronic central administration of KiSS-1 peptide (1 nmol per 12 h for 7 d) to pubertal males did not significantly modify relative expression levels of LHRH gene in whole hypothalamic/POA samples at 60 min after last injection (Fig. 3). Similarly, acute icv injection of 1 nmol KiSS-1 failed to induce significant changes in LHRH mRNA levels in whole hypothalamic/POA tissue over a period of 180 min (data not shown).
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). Likewise, iv administration of 7.5 nmol KiSS-1 elicited a potent LH secretory response in freely moving male rats. Thus, analysis of individual profiles of LH secretion demonstrated peak responses at 15 to 30 min after iv administration of KiSS-1 and a significant decline in circulating LH values thereafter. Accordingly, the integrated LH secretion during the 240-min period of sampling, as estimated by the AUC, was significantly increased by iv injection of 7.5 nmol KiSS-1 (Fig. 5).
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). Similarly, the potent LH secretory response to icv injection of KiSS-1 was persistently detected after pretreatment with the antagonist of NOSs NAME (Fig. 7B).
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Discussion |
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In this context, our current data document the extraordinarily potent LH-releasing activity of centrally administered KiSS-1 peptide, in keeping with our initial observations on the effects of icv injection of 1 nmol KiSS-1 (16). Thus, dose-response analyses evidenced that an input as low as 10 pmol KiSS-1 was able to maximally activate LH secretion to a level similar to that induced by systemic administration of LHRH, 15 min after injection. Although a certain degree of dose dependency was detected for the decay of the effects of KiSS-1 on serum LH levels over a 60-min period, it becomes evident by comparison with previously published data on the LH-releasing activity of other neuropeptides and neurotransmitters, such as glutamate and galanin-like peptide (28, 29), that the biopotency of KiSS-1 is considerably higher than that of other known regulators of LH secretion. In fact, during the final stage of preparation of this manuscript, Gottsch et al. (17) independently reported a detailed dose-response analysis of the effect of a wide range of doses of KiSS-1 peptide (1 fmol to 5 nmol) on LH secretion in the mouse. Although a single (30 min) time point was screened at that study, those data fully support our present observations and further extend the characterization of the extraordinarily potent LH-releasing activity of KiSS-1 peptide because a dose as low as 1 fmol of kisspeptin-54 was able to significantly increase serum LH levels (17).
The mechanisms whereby central administration of KiSS-1 peptide maximally activates LH secretion were addressed by a combination of approaches, targeting the putative contribution of hypothalamic LHRH to such a phenomenon. Our results demonstrate that, despite the use of maximally effective doses of KiSS-1 peptide, its ability to centrally elicit LH secretion is totally abrogated in the absence of endogenous LHRH actions in the male, in keeping with our recent observations in immature female rats (Navarro, V. M., and M. Tena-Sempere, submitted material). In addition, icv injection of KiSS-1 equaled the maximum stimulation of LH secretion induced by systemic administration of LHRH, but such a response could not be further increased by the combined administration of LHRH (ip) and KiSS-1 (icv). Although we cannot rule out the possibility that maximum LH responses were obtained at the doses used for each individual peptide, such a lack of additivity indirectly points out the convergence of KiSS-1 and LHRH pathways in a level upstream of the LH-secreting cells. Overall, our data strongly suggest that the effects of central KiSS-1 on LH secretion are mainly conducted through modulation of LHRH system. In this sense, GPR54 gene expression has been very recently demonstrated in LHRH neurons from the nonmammalian species tilapia (Oreochromis niloticus; cichlid fish) (30). Similarly, our preliminary data evidence that GPR54 gene is also expressed in the murine LHRH neuronal cell line GT1–7 (Magni, P., and M. Tena-Sempere, manuscript in preparation). Thus, it is tempting to propose that KiSS-1 peptide, acting through its receptor GPR54, is able to directly elicit the secretory activity of hypothalamic LHRH neurons. Although the precise mechanisms whereby this action is eventually conducted are yet to be identified, it appears likely that the potent LH-releasing effect of KiSS-1 is not due to the transcriptional activation of LHRH gene, as evidenced by our expression data of LHRH mRNA levels after icv injection of highly effective doses of KiSS-1. Nevertheless, the possibility of nucleus-specific effects of KiSS-1 on LHRH gene expression in a subset of LHRH neurons cannot be excluded on the basis of our current data. Alternatively, KiSS-1 might selectively stimulate LHRH release, in keeping with indirect evidence obtained in mouse models bearing mutations of GPR54 gene (14). This phenomenon is presently under evaluation at our laboratory.
Despite solid evidence pointing to a central hypothalamic system involving KiSS-1 and LHRH in the control of LH secretion, not only intracerebroventricular but also systemic (ip and iv) administration of KiSS-1 was able to significantly activate LH secretion. In fact, the effect of peripheral ip administration of KiSS-1 was similar in terms of maximum mean response to that of central injection of the peptide. Moreover, iv injection of KiSS-1 to freely moving animals sharply elicited a massive LH pulse in every single animal tested (see Fig. 5
). Several mechanisms may account for such a phenomenon. First, it is likely that systemically delivered KiSS-1 may regulate LHRH release by LHRH neuron nerve terminals located at the median eminence-arcuate nucleus complex, which is placed outside the blood-brain barrier (31). In this sense, expression of KiSS-1 gene has been very recently demonstrated in several hypothalamic areas, including the arcuate nucleus (17), thus evidencing the potential interaction between KiSS-1 and LHRH neurons at this location. In addition, however, it is possible that part of the stimulatory action of systemic KiSS-1 may derive from a direct releasing effect at the pituitary level because dose-dependent stimulation of LH secretion by incubated pituitary tissue was observed after challenge with increasing concentrations of KiSS-1. This would imply that, regardless of its predominant central effect in the control of the LHRH-LH axis, hypothalamic KiSS-1 might also operate as true hypophysiotropic factor involved in the control of LH secretion at the pituitary level. Notably, expression of GPR54 gene has been demonstrated at the pituitary (3); yet its pattern of cellular distribution and regulation by hormonal signals and at different stages of development remain to be elucidated in this tissue.
The relative contribution of direct pituitary effects to the potent releasing activity of KiSS-1 is likely minor (equimolar doses of KiSS-1 were 4 times less potent than 10–8 M LHRH in vitro, whereas systemic administration of KiSS-1 was as effective as LHRH in vivo) and would require the permissive presence of LHRH. Yet KiSS-1 was able to moderately elicit LH secretion in vitro at dose as low as 10–10 M, i.e. in a range that is close to the threshold levels for LHRH-induced LH stimulation (our personal observation); a phenomenon whose relevance merits further investigation. Nevertheless, from a functional standpoint, our current results document the potent LH-releasing ability of systemically delivered KiSS-1 peptides, which may have both physiological and therapeutical implications. On the former, it is noticeable that KiSS-1 peptides, such as metastin, are detected in plasma, and their concentrations dramatically increase under certain physiological conditions, such as pregnancy (12).
Considering that our experimental data suggested a predominant role of central KiSS-1/LHRH network in the control of LH secretion, we aimed at evaluating the potential interplay between KiSS-1 and other relevant neurotransmitters previously implicated in the neuronedocrine modulation of LH release, such as EAAs (glutamate) and NO (27, 32). In this sense, a prevalent role of hypothalamic glutamatergic pathways in the pubertal activation of the LHRH-LH axis has been demonstrated (27, 28), and a role for NO as a synchronizing factor for the disperse population of LHRH neurons has been proposed (32). Our results using in vivo models of pharmacological blockade of ionotropic EAA receptors of the NMDA and non-NMDA type as well as inhibition of NOSs in prepubertal male rats demonstrate that the integrity of EAA and NO neurotransmission is not needed for the expression of the potent LH-releasing ability of centrally administered KiSS-1. These observations suggest that KiSS-1 is placed in a step distal to (or eventually independent of) glutamate and NO actions within the central circuitry governing LHRH release. An interesting possibility is that those neurotransmitters might regulate the secretory activity of LHRH neurons by primary modulating the central expression of KiSS-1. Such a possibility is currently under analysis at our laboratory.
In summary, our results provide conclusive evidence for a potent stimulatory effect of KiSS-1 peptide on LH secretion. Such a releasing action is mostly conducted centrally, likely through modulation of the LHRH system. However, the contribution of stimulatory effects of KiSS-1 on LH secretion directly at the pituitary level cannot be excluded. Overall, the present results, in conjunction with our recent data on the developmental and hormonally regulated pattern of expression of KiSS-1 and GPR54 genes in the hypothalamus (16), and their potential involvement in the activation of the reproductive axis at puberty (Navarro, V. M., and M. Tena-Sempere, submitted material), further document a novel role of the KiSS-1/GPR54 system as a relevant downstream element in the neuroendocrine network governing LH secretion.
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Acknowledgments |
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Footnotes |
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First Published Online September 16, 2004
1 V.M.N. and J.M.C. equally contributed to this work and should be considered as joint first authors. 
Abbreviations: AMPA, 2-Amino-3-hydroxy-5-methyl-4-isoxazol propionic acid; AUC, area under the curve; EAA, excitatory amino acid; icv, intracerebroventricular; KA, kainate; NAME, N-Nitrow-arginine-methyl ester; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, NO synthase; POA, preoptic area; RT, reverse transcription.
Received July 1, 2004.
Accepted for publication September 9, 2004.
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-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in infantile, prepubertal, and adult male rats. Endocrinology 140:1279–1284This article has been cited by other articles:
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 H. M. Dungan, M. L. Gottsch, H. Zeng, A. Gragerov, J. E. Bergmann, D. K. Vassilatis, D. K. Clifton, and R. A. Steiner The Role of Kisspeptin GPR54 Signaling in the Tonic Regulation and Surge Release of Gonadotropin-Releasing Hormone/Luteinizing Hormone J. Neurosci., October 31, 2007; 27(44): 12088 - 12095. [Abstract] [Full Text] [PDF]
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 W. S. Dhillo, O. B. Chaudhri, E. L. Thompson, K. G. Murphy, M. Patterson, R. Ramachandran, G. K. Nijher, V. Amber, A. Kokkinos, M. Donaldson, et al. Kisspeptin-54 Stimulates Gonadotropin Release Most Potently during the Preovulatory Phase of the Menstrual Cycle in Women J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 3958 - 3966. [Abstract] [Full Text] [PDF]
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D. Li, D. Mitchell, J. Luo, Z. Yi, S.-G. Cho, J. Guo, X. Li, G. Ning, X. Wu, and M. Liu Estrogen Regulates KiSS1 Gene Expression through Estrogen Receptor {alpha} and SP Protein Complexes Endocrinology, October 1, 2007; 148(10): 4821 - 4828. [Abstract] [Full Text] [PDF]
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 D. Marot, I. Bieche, C. Aumas, S. Esselin, C. Bouquet, S. Vacher, G. Lazennec, M. Perricaudet, F. Kuttenn, R. Lidereau, et al. High tumoral levels of Kiss1 and G-protein-coupled receptor 54 expression are correlated with poor prognosis of estrogen receptor-positive breast tumors Endocr. Relat. Cancer, September 1, 2007; 14(3): 691 - 702. [Abstract] [Full Text] [PDF]
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 B. J Todd, G. S Fraley, A. C Peck, G. J Schwartz, and A. M Etgen Central Insulin-Like Growth Factor 1 Receptors Play Distinct Roles in the Control of Reproduction, Food Intake, and Body Weight in Female Rats Biol Reprod, September 1, 2007; 77(3): 492 - 503. [Abstract] [Full Text] [PDF]
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A. S. Kauffman, M. L. Gottsch, J. Roa, A. C. Byquist, A. Crown, D. K. Clifton, G. E. Hoffman, R. A. Steiner, and M. Tena-Sempere Sexual Differentiation of Kiss1 Gene Expression in the Brain of the Rat Endocrinology, April 1, 2007; 148(4): 1774 - 1783. [Abstract] [Full Text] [PDF]
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Y. Tenenbaum-Rakover, M. Commenges-Ducos, A. Iovane, C. Aumas, O. Admoni, and N. de Roux Neuroendocrine Phenotype Analysis in Five Patients with Isolated Hypogonadotropic Hypogonadism due to a L102P Inactivating Mutation of GPR54 J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 1137 - 1144. [Abstract] [Full Text] [PDF]
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T. J. Greives, A. O. Mason, M.-A. L. Scotti, J. Levine, E. D. Ketterson, L. J. Kriegsfeld, and G. E. Demas Environmental Control of Kisspeptin: Implications for Seasonal Reproduction Endocrinology, March 1, 2007; 148(3): 1158 - 1166. [Abstract] [Full Text] [PDF]
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E. J. Mead, J. J. Maguire, R. E. Kuc, and A. P. Davenport Kisspeptins Are Novel Potent Vasoconstrictors in Humans, with a Discrete Localization of Their Receptor, G Protein-Coupled Receptor 54, to Atherosclerosis-Prone Vessels Endocrinology, January 1, 2007; 148(1): 140 - 147. [Abstract] [Full Text] [PDF]
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J. M. Castellano, V. M. Navarro, R. Fernandez-Fernandez, J. Roa, E. Vigo, R. Pineda, R. A. Steiner, E. Aguilar, L. Pinilla, and M. Tena-Sempere Effects of galanin-like peptide on luteinizing hormone secretion in the rat: sexually dimorphic responses and enhanced sensitivity at male puberty Am J Physiol Endocrinol Metab, December 1, 2006; 291(6): E1281 - E1289. [Abstract] [Full Text] [PDF]
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W. S. Dhillo, P. Savage, K. G. Murphy, O. B. Chaudhri, M. Patterson, G. M. Nijher, V. M. Foggo, G. S. Dancey, H. Mitchell, M. J. Seckl, et al. Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E878 - E884. [Abstract] [Full Text] [PDF]
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E. L. Thompson, K. G. Murphy, M. Patterson, G. A. Bewick, G. W. H. Stamp, A. E. Curtis, J. H. Cooke, P. H. Jethwa, J. F. Todd, M. A. Ghatei, et al. Chronic subcutaneous administration of kisspeptin-54 causes testicular degeneration in adult male rats Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E1074 - E1082. [Abstract] [Full Text] [PDF]
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 J. M. Castellano, V. M. Navarro, R. Fernandez-Fernandez, J. Roa, E. Vigo, R. Pineda, C. Dieguez, E. Aguilar, L. Pinilla, and M. Tena-Sempere Expression of Hypothalamic KiSS-1 System and Rescue of Defective Gonadotropic Responses by Kisspeptin in Streptozotocin-Induced Diabetic Male Rats Diabetes, September 1, 2006; 55(9): 2602 - 2610. [Abstract] [Full Text] [PDF]
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M. Tena-Sempere GPR54 and kisspeptin in reproduction Hum. Reprod. Update, September 1, 2006; 12(5): 631 - 639. [Abstract] [Full Text] [PDF]
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S. Tovar, M. J. Vazquez, V. M. Navarro, R. Fernandez-Fernandez, J. M. Castellano, E. Vigo, J. Roa, F. F. Casanueva, E. Aguilar, L. Pinilla, et al. Effects of Single or Repeated Intravenous Administration of Kisspeptin upon Dynamic LH Secretion in Conscious Male Rats Endocrinology, June 1, 2006; 147(6): 2696 - 2704. [Abstract] [Full Text] [PDF]
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J. Roa, E. Vigo, J. M. Castellano, V. M. Navarro, R. Fernandez-Fernandez, F. F. Casanueva, C. Dieguez, E. Aguilar, L. Pinilla, and M. Tena-Sempere Hypothalamic Expression of KiSS-1 System and Gonadotropin-Releasing Effects of Kisspeptin in Different Reproductive States of the Female Rat Endocrinology, June 1, 2006; 147(6): 2864 - 2878. [Abstract] [Full Text] [PDF]
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 V. M. Navarro, R. Fernandez-Fernandez, R. Nogueiras, E. Vigo, S. Tovar, N. Chartrel, O. Le Marec, J. Leprince, E. Aguilar, L. Pinilla, et al. Novel role of 26RFa, a hypothalamic RFamide orexigenic peptide, as putative regulator of the gonadotropic axis J. Physiol., May 15, 2006; 573(1): 237 - 249. [Abstract] [Full Text] [PDF]
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A. C. Martini, R. Fernandez-Fernandez, S. Tovar, V. M. Navarro, E. Vigo, M. J. Vazquez, J. S. Davies, N. M. Thompson, E. Aguilar, L. Pinilla, et al. Comparative Analysis of the Effects of Ghrelin and Unacylated Ghrelin on Luteinizing Hormone Secretion in Male Rats Endocrinology, May 1, 2006; 147(5): 2374 - 2382. [Abstract] [Full Text] [PDF]
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S. B. Seminara, M. J. DiPietro, S. Ramaswamy, W. F. Crowley Jr., and T. M. Plant Continuous Human Metastin 45-54 Infusion Desensitizes G Protein-Coupled Receptor 54-Induced Gonadotropin-Releasing Hormone Release Monitored Indirectly in the Juvenile Male Rhesus Monkey (Macaca mulatta): A Finding with Therapeutic Implications Endocrinology, May 1, 2006; 147(5): 2122 - 2126. [Abstract] [Full Text] [PDF]
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J. T Smith, D. K Clifton, and R. A Steiner Regulation of the neuroendocrine reproductive axis by kisspeptin-GPR54 signaling. Reproduction, April 1, 2006; 131(4): 623 - 630. [Abstract] [Full Text] [PDF]
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 J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]
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S. R. Ojeda, A. Lomniczi, C. Mastronardi, S. Heger, C. Roth, A.-S. Parent, V. Matagne, and A. E. Mungenast Minireview: The Neuroendocrine Regulation of Puberty: Is the Time Ripe for a Systems Biology Approach? Endocrinology, March 1, 2006; 147(3): 1166 - 1174. [Abstract] [Full Text] [PDF]
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H. M. Dungan, D. K Clifton, and R. A. Steiner Minireview: Kisspeptin Neurons as Central Processors in the Regulation of Gonadotropin-Releasing Hormone Secretion Endocrinology, March 1, 2006; 147(3): 1154 - 1158. [Abstract] [Full Text] [PDF]
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S. Pompolo, A. Pereira, K. M. Estrada, and I. J. Clarke Colocalization of Kisspeptin and Gonadotropin-Releasing Hormone in the Ovine Brain Endocrinology, February 1, 2006; 147(2): 804 - 810. [Abstract] [Full Text] [PDF]
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T. M. Plant, S. Ramaswamy, and M. J. DiPietro Repetitive Activation of Hypothalamic G Protein-Coupled Receptor 54 with Intravenous Pulses of Kisspeptin in the Juvenile Monkey (Macaca mulatta) Elicits a Sustained Train of Gonadotropin-Releasing Hormone Discharges Endocrinology, February 1, 2006; 147(2): 1007 - 1013. [Abstract] [Full Text] [PDF]
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S.-K. Han, M. L. Gottsch, K. J. Lee, S. M. Popa, J. T. Smith, S. K. Jakawich, D. K. Clifton, R. A. Steiner, and A. E. Herbison Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty J. Neurosci., December 7, 2005; 25(49): 11349 - 11356. [Abstract] [Full Text] [PDF]
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W. S. Dhillo, O. B. Chaudhri, M. Patterson, E. L. Thompson, K. G. Murphy, M. K. Badman, B. M. McGowan, V. Amber, S. Patel, M. A. Ghatei, et al. Kisspeptin-54 Stimulates the Hypothalamic-Pituitary Gonadal Axis in Human Males J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6609 - 6615. [Abstract] [Full Text] [PDF]
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 A. C. Arai, Y.-F. Xia, E. Suzuki, M. Kessler, O. Civelli, and H.-P. Nothacker Cancer Metastasis-Suppressing Peptide Metastin Upregulates Excitatory Synaptic Transmission in Hippocampal Dentate Granule Cells J Neurophysiol, November 1, 2005; 94(5): 3648 - 3652. [Abstract] [Full Text] [PDF]
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M. Tena-Sempere Hypothalamic KiSS-1: The Missing Link in Gonadotropin Feedback Control? Endocrinology, September 1, 2005; 146(9): 3683 - 3685. [Full Text] [PDF]
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J. T. Smith, M. J. Cunningham, E. F. Rissman, D. K Clifton, and R. A. Steiner Regulation of Kiss1 Gene Expression in the Brain of the Female Mouse Endocrinology, September 1, 2005; 146(9): 3686 - 3692. [Abstract] [Full Text] [PDF]
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J. M. Castellano, V. M. Navarro, R. Fernandez-Fernandez, R. Nogueiras, S. Tovar, J. Roa, M. J. Vazquez, E. Vigo, F. F. Casanueva, E. Aguilar, et al. Changes in Hypothalamic KiSS-1 System and Restoration of Pubertal Activation of the Reproductive Axis by Kisspeptin in Undernutrition Endocrinology, September 1, 2005; 146(9): 3917 - 3925. [Abstract] [Full Text] [PDF]
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J. T. Smith, H. M. Dungan, E. A. Stoll, M. L. Gottsch, R. E. Braun, S. M. Eacker, D. K Clifton, and R. A. Steiner Differential Regulation of KiSS-1 mRNA Expression by Sex Steroids in the Brain of the Male Mouse Endocrinology, July 1, 2005; 146(7): 2976 - 2984. [Abstract] [Full Text] [PDF]
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S. B. Seminara and U. B. Kaiser New Gatekeepers of Reproduction: GPR54 and Its Cognate Ligand, KiSS-1 Endocrinology, April 1, 2005; 146(4): 1686 - 1688. [Full Text] [PDF]
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V. M. Navarro, J. M. Castellano, R. Fernandez-Fernandez, S. Tovar, J. Roa, A. Mayen, M. L. Barreiro, F. F. Casanueva, E. Aguilar, C. Dieguez, et al. Effects of KiSS-1 Peptide, the Natural Ligand of GPR54, on Follicle-Stimulating Hormone Secretion in the Rat Endocrinology, April 1, 2005; 146(4): 1689 - 1697. [Abstract] [Full Text] [PDF]
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 M. Shahab, C. Mastronardi, S. B. Seminara, W. F. Crowley, S. R. Ojeda, and T. M. Plant Increased hypothalamic GPR54 signaling: A potential mechanism for initiation of puberty in primates PNAS, February 8, 2005; 102(6): 2129 - 2134. [Abstract] [Full Text] [PDF]
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